South African Journal of Botany 132 (2020) 471�481

Contents lists available at ScienceDirect

South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Burrowsia, a new genus of lichenized fungi (Caliciaceae), plus the newspecies B. cataractae and Scoliciosporum fabisporum, from Mpumalanga,

South Africa

Alan M. Frydaya,*, Ian D. Medeirosb,c, Stefan J. Siebertd, Nathaniel Popee,Nishanta Rajakarunac,d,f

aHerbarium, Department Plant Biology, Michigan State University, East Lansing, MI 48823, USAb Department of Biology, Duke University, Durham, NC 27708, USAc College of the Atlantic, 105 Eden Street, Bar Harbor, ME 04609, USAd Unit for Environmental Sciences and Management, North-West University, Potchefstroom 2520, South AfricaeDepartment of Entomology, Pennsylvania State University, University Park, PA 16802, USAf Biological Sciences Department, California Polytechnic State University, San Luis Obispo, CA 93407, USA

A R T I C L E I N F O

Article History:Received 15 January 2020Accepted 1 June 2020Available online xxx

Edited by J Van Staden

* Corresponding author.E-mail address: fryday@msu.edu (A.M. Fryday).

https://doi.org/10.1016/j.sajb.2020.06.0010254-6299/© 2020 SAAB. Published by Elsevier B.V. All r

A B S T R A C T

The new genus Burrowsia (Caliciaceae) is proposed to accommodate the new species B. cataractae, which isknown from only a single locality in Mpumalanga, South Africa. Burrowsia is characterized by its pigmented,submuriform ascospores and ascus with an apical tube structure, and also by its DNA sequence data thatplace it outside related buellioid genera. We also describe the new species Scoliciosporum fabisporum, alsoknown from a single locality in Mpumalanga, which differs from all other species of that genus in having dis-tinctive kidney-shaped, 0�1-septate ascospores. It is most closely related to the Northern Hemisphere spe-cies S. intrusum, which is here confirmed by molecular data as belonging to this genus in a well-supportedScoliciosporaceae. The potential of the region to yield additional novel lichen taxa is explored.

© 2020 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords:

Endemic speciesLichensNew taxaScoliciosporaceaeUltramafic rocks

ights reserved.

1. Introduction

Lichens are an integral part of most terrestrial ecosystems, fromarid polar deserts to tropical rainforests, from maritime rocks toalpine summits. They provide important ecosystem services such asnutrient recycling, food and shelter for many species of animals (e.g.,birds, snails, ants and mites), regulate soil moisture and producenitrogen for other species (e.g., Asplund and Wardle 2013, 2017;Elbert et al., 2012; Knops et al., 1996; Slack 1988). Unfortunately,they are often overlooked and their biodiversity and ecology arepoorly known or understood in many parts of the world. This is par-ticularly true of crustose lichens that typically constitute around two-thirds of all lichen biodiversity in an area (Lendemer et al., 2013;Spribille et al., 2020).

The lichen biota of South Africa is reasonably well documented.The current checklist (Fryday, 2015 and 2016; Ahti et al., 2016) lists 1751 taxa, and a second supplement currently in preparation(Medeiros and Fryday, in prep.) adds a further 15 taxa, making a total

of 1 766. Estimates for the potential total number of taxa present inthe country range from 2 000 (Crous et al., 2006) to 3 000 (Fry-day, 2015), which is well below the over 21 000 vascular plantsreported for the country (Schnitzler et al., 2011) and low when com-pared with the 1 838 taxa reported from Great Britain (Smith et al.,2009), a country which is only one-fifth the size. At the same time,the lichen biodiversity is poorly understood — especially the microli-chen biota (crustose species). Many of these were described fromSouth Africa in the late 19th century and are known only from theirtype collections, which have often not been studied since the specieswere described. Chief among these are the taxa described by ErnestusStizenberger in his Lichenaea Africana (Stizenberger, 1890 and 1891),but Nylander (1869 and in Crombie, 1876), Vainio (1926) and Zahl-bruckner (1906, 1926, 1932 and 1936) also described many new spe-cies from the country. Since that time, there has been intermittentresearch on the lichen biota of South Africa, mainly by visiting Euro-pean lichenologists (e.g., Almborn, 1966 and 1987; Schultz et al.,2009) but also by researchers resident in South Africa, most notablyFranklin Brusse. Between 1985 and 1994, Brusse published over 30papers describing new species from the country and often includednew reports of other species (see Fryday, 2015 for references and a

472 A.M. Fryday et al. / South African Journal of Botany 132 (2020) 471�481

fuller history of lichenology in South Africa). However, theseresearchers have usually restricted themselves to describing newspecies and have not explored the systematic position or relation-ships of their discoveries (but see Aptroot et al., 2019;Kondratyuk et al., 2015; Leavitt et al., 2018a and b). Here we describetwo new species of saxicolous, crustose lichens— the group of lichensthat suffers from the greatest lack of attention within the South Afri-can lichen biota (Fryday, 2015) — that are known only from SouthAfrica. One of these species is shown to occupy an isolated positionwithin the Caliciaceae on the basis of both molecular and morpholog-ical data and so we erect a new genus to accommodate it. The otherspecies is readily accommodated within the genus Scoliciosporum A.Massal.

2. Materials and methods

This study is based upon specimens of crustose, lecideoid lichenscollected by the first author from Mpumalanga, South Africa, in 2015and 2016.

2.1. Morphological methods

Apothecial characteristics were examined by light microscopy onhand-cut sections mounted in water, 10% KOH (K), 50% HNO3 (N) orLugol’s reagent (0.15% aqueous IKI). Thallus sections were investigated inwater, K and lactophenol cotton-blue. The ascus structure was studied inIKI, both without prior treatment and after pretreatment with K. Meas-urements of ascospores and paraphyses were made in K. Ascosporedimensions are given as (smallest measured�) arithmetic mean§stan-dard deviation (�largest measured). Hamathecial filaments are referredto as ‘paraphyses’ regardless of their origin. Thin layer chromatography(TLC) follows the methods of Orange et al., 2001 . Nomenclature for apo-thecial pigments followsMeyer and Printzen, 2000.

2.2. Molecular methods

Approximately 10 apothecia from each of the two holotype speci-mens were excised from the thallus, frozen with liquid nitrogen, andground to a powder with a plastic pestle. Pulverized samples werethen lysed overnight in a 2% sodium dodecyl sulfate (SDS) extractionbuffer. DNA extraction followed a standard phenol:chloroform proto-col. Extracted DNA was precipitated with the addition of isopropylalcohol and centrifugation before being cleaned with 80% ethanoland a final centrifugation. The DNA pellet was air-dried before beingrehydrated in sterile water and stored at �20 °C.

We attempted to amplify the following regions from the mycobiontgenome with polymerase chain reaction (PCR): the mitochondrial smallsubunit (mtSSU) using the primer pair mrSSU1 and mrSSU3R(Zoller et al., 1999); the internal transcribed spacer (ITS; including ITS1,5.8S, and ITS2) using the primer pair ITS1F (Gardes and Bruns, 1993) andITS4 (White et al., 1990) or LR3 (Vilgalys and Hester, 1990); the nuclearlarge subunit (nrLSU) using the primer pair LR0R (Rehner andSamuels, 1994) and LR7 (Vilgalys and Hester, 1990); the nuclear smallsubunit (nrSSU) using the primer pair NSSU131 (Kauff and Lutzoni, 2002)and NS24 (Gargas and Taylor, 1992); the RNA polymerase II largest sub-unit (RPB1) using the primer pair RPB1-aFasc (Hofstetter et al., 2007) andRPB1-cR (Matheny et al., 2002); and the RNA polymerase II second-largestsubunit (RPB2) using the primer pair fRPB2�7cF and fRPB2�11aR(Liu et al., 1999). To identify the photobiont, we also amplified the algalITS region with primers ITS1T (Kroken and Taylor, 2000) and either ITS4or LR3. PCR products were viewed on 1% agarose gels and subjected to anenzymatic cleanup step with exonuclease and shrimp alkaline phospha-tase. Sanger sequencing was performed by Eurofins Genomics (ResearchTriangle Park, North Carolina, USA) using PCR primers, except that ITS1(White et al., 1990) was substituted for ITS1F. Forward and reverse readswere assembled in Sequencher 5.4.6 (Gene Codes Corporation, Ann

Arbor, MI, USA) or Geneious Prime 2020.0.3 (www.geneious.com) andthe assembled contigs were checked by eye for base call errors.

We used the online T-BAS portal (Carbone et al., 2016 and 2019;Miller et al., 2015) to obtain preliminary phylogenetic placements forthe two species. T-BAS uses the RAxML evolutionary placement algo-rithm (Berger et al., 2011) to infer approximate phylogenetic place-ments for input sequences. We placed the newly obtained sequencesin a phylogeny of Pezizomycotina to confirm their position in Leca-noromycetes, then re-ran the placement with a phylogeny of Leca-noromycetes (Miadlikowska et al., 2014) to determine a family-levelplacement. Sequences from related species were obtained from Gen-Bank, and additional new sequence data were generated for a speci-men of Pseudothelomma occidentale (Herre) M. Prieto & Wedin(Alaska, Fryday 10 069, MSC). ITS and mtSSU sequences were alignedwith MAFTT (Katoh et al., 2017; Kuraku et al., 2013) using the G-INS-1 method; alignment errors were corrected and ambiguously alignedregions were excluded by eye in Mesquite. Nuclear rDNA regionsnrLSU and nrSSU were aligned by eye in Mesquite according toknown intron positions. Protein-coding sequences (RPB1, RPB2, beta-tubulin and MCM7) were aligned in Mesquite using the amino acidtranslation. Intron sequences were excluded from the analysis. Sin-gle-locus trees were inferred with RAxML (Stamatakis, 2014 ); find-ing no well-supported conflicts in the single locus trees for either ofour species, we performed subsequent analyses on concatenateddatasets. PartitionFinder2 (Lanfear et al., 2016 ) was used to delimitpartitions in the concatenated alignment of all loci and determine thebest evolutionary model for the data. We ran Partitionfinder2 withthe greedy search algorithm, allowing independent branch lengthsper partition, and used the second-order Akaike Information Crite-rion (AICc) to determine the best-performing substitution model andpartition scheme. The partitioned datasets were subjected to maxi-mum likelihood analysis in RAxML 8.2.12 using the GTR+I+G model;RAxML was allowed to automatically halt bootstrapping with theautoMRE stopping criterion (Pattengale et al., 2009). PartitionFinder2and RAxML were run on the CIPRES server (Miller et al., 2010). Align-ment files and trees have been uploaded to TreeBASE as accessionS26468. To identify photobiont genus, we used BLAST (Zhang et al.,2000) to compare the algal ITS sequences against the NCBI Nucleotidedatabase.

3. Results

3.1. Molecular results

Morphological studies indicated that the two saxicolous speci-mens represent two species new to science. The preliminary analysisin T-BAS showed that sequences from the two specimens were con-sistently associated with Caliciaceae and Scoliciosporum intrusum (Th.Fr.) Hafellner (Lecanorales incertae sedis), respectively. Usingsequences from Miadlikowska et al. (2014) as a starting point,we assembled sequence datasets for Caliciaceae and Lecanorales,the latter focusing on clades outside suborder Lecanorineae(Miadlikowska et al., 2014). The five-locus dataset for Lecanorales(ITS, mtSSU, nrSSU, nrLSU, and RPB1; Table 1) includes 4520 charac-ters and is dominated by sequences from Andersen andEkman (2005), with additions from other studies. The eight-locusdataset for Caliciaceae (ITS, mtSSU, nrSSU, nrLSU, RPB1, RPB2, beta-tubulin and MCM7; Table 2) includes 9726 characters and containsmany sequences from Prieto and Wedin (2017). The maximum likeli-hood analysis for Caliciaceae (Fig. 1) confirmed the placement of oneof the new species in this family as a long branch among several buel-lioid genera, whereas the analysis of Lecanorales (Fig. 2) confirmedthe relationship of the other new species with Scoliciosporum intru-sum within a well-supported Scoliciosporaceae. Based on the mor-phological and molecular results, one new genus and two newspecies are described below.

Table 1GenBank accession numbers for specimens used in the phylogenetic analysis of Caliciaceae. Physciaceae (Heterodermia vulgaris + Physcia aipolia) was used asthe outgroup. Newly generated sequences are in bold. The final row shows the number of sites for each locus present in the concatenated alignment.

Species ITS mtSSU nrSSU nrLSU RPB1 RPB2 beta-tubulin MCM7

Physcia aipolia DQ782836 DQ912290 DQ782876 DQ782904 DQ782820 DQ782862 KX529021 KX529052Heterodermia vulgaris HQ650704 DQ912288 DQ883789 DQ883798 DQ883741 DQ883754 — —

Acolium inquinans AY450583 AY143404 U86695 AY453639 — — KX529023 —

Acolium karelicum KX512897 — — KX512879 — — — KX529045Acroscyphus sphaerophoroides KX512898 KX512984 — — — — — KX529029Allocalicium adaequatum KX512906 KX512986 — KX512859 — — KX528996 —

Amandinea coniops KJ607904 KX512978 — KX512865 — — KX528998 —

Amandinea punctata HQ650627 DQ986874 DQ986721 DQ986756 KJ766906 DQ992435 — KX529025Baculifera remensa — KX512962 — KX512881 — — — —

Buellia disciformis AY143392 AY143401 AF241543 AY340537 — — — —

Buellia elegans AY143411 AY143417 — — — — KX528988 —

Buellia erubescens KX512902 KX512969 — KX512874 — — KX529004 —

Buellia fimbriata — — DQ973010 DQ973034 DQ973057 DQ973097 — —

Buellia frigida HQ650628 DQ986903 DQ883699 DQ883695 DQ883724 DQ883712 — —

Buellia stillingiana — DQ912287 DQ912319 — DQ912368 DQ912391 — —

Buellia tesserata KX512904 — — KX512885 — — — KX529050Burrowsia cataractae MT622501 — — MT622322 MT610731 MT610733 — —

Calicium pinicola KX512917 KX512991 — KX512887 — — KX529014 KX529066Calicium tigillare AY452498 JQ301534 JQ301646 JQ301593 — — KX529002 JX000162Calicium viride HQ650703 AY584696 AF356669 AF356670 — AY641031 KX529013 —

Culbersonia nubila MH121318 — — MH121320 — — — —

Dimelaena radiata JQ301693 — JQ301647 JQ301594 JQ301736 JQ301787 — KX529049Diploicia canescens AF250793 AY464084 AJ421684 — — — — —

Diplotomma alboatrum KX512924 KX512966 — KX512877 — — KX529007 KX529043Diplotomma epipolium AF540509 JQ301535 AJ506969 JQ301595 — — — —

Pseudothelomma occidentale MT622500 — MT611985 MT611534 MT610734 MT610735 — —

Pseudothelomma ocellatum KF020559 JQ301540 — JQ301599 — — KX529020 KX529063Pyxine sorediata JQ301697 DQ972984 DQ973012 DQ973036 — DQ973071 KX529001 KX529039Pyxine subcinerea HQ650705 DQ912292 DQ883793 DQ883802 DQ883745 DQ883758 — —

Tetramelas chloroleucus KX512938 — — KX512875 — — KX529006 —

Texosporium sancti-jacobi KX512941 KX512981 — KX512863 — — KX528994 KX529031Thelomma mammosum KX512943 KX512953 — KX512851 — — KX529017 KX529067Thelomma santessonii KX512944 KX512951 — KX512889 — — — KX529064Tholurna dissimilis AY452499 DQ972974 DQ973002 — — — KX528992 KX529053Alignment sites 264 690 1582 1304 2760 1881 687 558

A.M. Fryday et al. / South African Journal of Botany 132 (2020) 471�481 473

3.2. The taxa

Burrowsia Fryday and I. Medeiros gen. nov.Mycobank No.: MB 835878The new genus is characterized by its pigmented, submuriform

ascospores and asci with an apical tube-like structure. It is furtherdistinguished from all other genera by its DNA sequence data.

Type species: Burrowsia cataractae Fryday and I. MedeirosEtymology: The name honours John and Sandra (Sandie) Burrows,

the managers of Buffelskloof Nature Reserve from where the typespecies was collected, for their outstanding, life-long contribution toconservation and biodiversity research in South Africa.

Because the genus is monotypic, a separate description of thegenus would be superfluous.

Burrowsia cataractae Fryday and I. Medeiros sp. nov.Mycobank No.: MB 835879Separated from all other species in the Caliciaceae by its submuri-

form ascospores, ascus structure and DNA sequence data. Superfi-cially similar to Rhizocarpon lavatum but with a different ascusstructure, smaller pigmented submuriform ascospores and a thalluscontaining norstictic acid and two unidentified substances.

Typus: South Africa, Mpumalanga, Ehlanzeni District, ThabaChweu Municipality, Mashishing (Lydenburg), Buffelskloof NatureReserve, Calodendrum Falls, 25° 17.8390S, 30° 30.6560E, 1500 m,damp closed forest, saxicolous boulders in splash zone of waterfall,11 Febuary 2016, A.M. Fryday (11 591), I. Medeiros, J. Burrows and A.Frisby (PRE—holo!) (Fig. 3).

Thallus widespreading, grey�brown (greenish�brown whenwet), rimose to cracked�areolate, margin obscurely lobed with a thinblack prothallus, 0.3�0.4 mm thick; upper surface layer §hyaline,

cells with dilutely brown pigmented cap buried within an epinecrallayer c. 10 mm deep; photobiont layer continuous, 20�70 mm deep;photobiont Trebouxia; cells chlorococcoid §orbicular 6�7mm diam. toelongate 10�12 £ 5�6mm; lower surface layer, c. 250 mm deep,brown pigmented, composed of §vertically aligned paraplecten-chyma, cells 10�15 £ 5�6mm.

Apothecia frequent, lecideine, orbicular, occasionally flexuosewhen mature, dark brown to black, semi�immersed to adnatewith a broad base, becoming sessile, (0.4�)0.6�0.8 mm diam.Proper margin c. 0.1 mm broad, slightly raise and persistent, palerthan the disk; in section cupular, initially brown but clearing andproducing red, acicular crystals with K, in thin section with abrown, poorly defined cortex and §hyaline medulla; composed ofrows of radiating hyphae, 6�10£ 4�5 mm. Hymenium110�130 mm, I+ blue; paraphyses §simple, rarely branched, sep-tate, becoming §moniliform above, 1.5�2.5 mm thick, widening to3�4 mm at apex with a thin brown cap. Ascus cylindrical,c. 70 £ (15�)20 mm, walls 2�3 mm thick; tholus IKI+ pale bluewith a central, darker-staining tube-like structure narrowingtowards the apex; ascospores 4�6(�8)/ascus, pigmented, submuri-form, non�halonate, (17�)23.61§4.37(�32) £ (7�)11.74§5.66(�15) mm, l/b ration (1.267�)2.05§0.39(�2.46), n = 23, ellipsoid,sometimes slightly curved. Hypothecium dark brown, c. 150mmthick, composed of §randomly aligned hyphae.

Conidiomata pycnidia, immersed in thallus with just dark ostiolevisible, flask�shaped with pale brown wall; conidia long bacilliform,10�12 £ 1mm.

Chemistry K+ red, C�, Pd�; norstictic acid and two unidentified sub-stances by TLC. Both unidentified substances gave UVC++ pinkish-creamspots; a large one at Rf 7.0 and a smaller one at Rf 5.5 in solvent C.

Table 2GenBank accession numbers for specimens used in the phylogenetic analysis of Lecanorales. Rusavskia ele-gans was used as the outgroup. Newly generated sequences are in bold. The final row shows the number ofsites for each locus present in the concatenated alignment.

Species ITS mtSSU nrSSU nrLSU RPB1

Rusavskia elegans EF423390 DQ912304 DQ912329 DQ912352 DQ973068Aquacidia antricola — MH817969 — — —

Bapalmuia palmularis AY756457 AY567781 — — —

Bilimbia sabuletorum — KJ766361 KJ766694 KJ766534 KJ766839Brianaria sylvicola JX983583 JX983587 — — AY756392

Byssoloma leucoblepharum AY756459 AY567778 AF455135 AY756317 AY756380Byssoloma subdiscordans AY756461 AY567779 KJ766696 KJ766538 KJ766841Calopadia foliicola AY756462 AY567782 — AY756318 AY756381Calopadia phyllogena — KJ766365 — KJ766539 —

Calycidium cuneatum JX000114 JX000117 — JX000083 JX000134Fellhanera bouteillei AF414858 KJ766392 KJ766716 KJ766559 —

Fellhanera subtilis AY756464 AY567786 — AY756321 —

Fellhaneropsis vezdae — AY567744 — — —

Lasioloma arachnoideum AY756467 AY567783 — — —

Lecanora hybocarpa DQ782849 DQ912273 DQ782883 DQ782910 DQ782829Leimonis erratica AY756475 AY567737 — AY756328 AY756390Micarea adnata AY756468 AY567751 AF455134 AY756326 AY756388Micarea alabastrites AY756469 AY567764 — AY756327 AY756389Micarea byssacea AY756485 AY567749 — AY756330 —

Micarea cinerea AY756472 AY567763 — — —

Micarea denigrata — KJ766437 KJ766750 KJ766598 KJ766873Micarea doliiformis HQ650654 HQ660557 — HQ660534 —

Micarea lapillicola AY756479 AY567735 — — —

Micarea lithinella AY756482 AY567734 — — —

Micarea misella AY756486 AY567752 — — —

Micarea myriocarpa AY756487 AY567736 — — —

Micarea paratropa AY756490 AY567740 — — —

Micarea synotheoides AY756493 AY567756 — — —

Protoblastenia calva HQ650618 DQ98690 JQ301653 JQ301601 DQ986830Protoblastenia rupestris — — KJ766771 KJ766631 KJ766880Psilolechia leprosa AY756496 AY567730 — AY756333 AY756395Psilolechia lucida — KJ766472 KJ766777 KJ766639 KJ766884Szczawinskia tsugae AY756499 — — — —

Scoliciosporum chlorococcum — AY567768 — — —

Scoliciosporum fabisporum — MT611934 — MT611533 MT610732

Scoliciosporum intrusum — AY567767 — AY756329 —

Scoliciosporum umbrinum — AY300911 AF091587 AY300861 AY756397Septotrapelia usnicum — AY300894 — AY300843 DQ870952Sphaerophorus fragilis HQ650600 DQ986789 DQ983487 DQ986805 —

Sphaerophorus globosus HQ650622 AY584723 DQ986712 DQ986767 DQ986836Sporopodium antoninianum AY756498 AY567785 — — —

Tephromela atra HQ650608 DQ986879 DQ986724 DQ986766 DQ986835Alignment sites 258 741 1601 1323 597

474 A.M. Fryday et al. / South African Journal of Botany 132 (2020) 471�481

Etymology: The specific epithet references the habitat of the onlyknown collection, which is at the base of a waterfall: cataractae = ofthe waterfall (Latin: genitive, singular).

Distribution and Ecology: The new species is known only from thetype collection, which is at the base of a waterfall in a deep ravine(Fig. 4). The falls are on the Dwaalheuvel Quartzites and the new spe-cies grows on moist upper surfaces of flat, quartzite rocks aroundrock pools where it would have been permanently damp, if not peri-odically inundated.

The vegetation of the ravine is classified as Eastern Dry Afrotem-perate Forest (L€otter et al., 2014). This forest type receives meanannual precipitation of 1 084 mm and has a mean annual tempera-ture of 16.7°C, but this is of little relevance to the highly humidmicrohabitat in which the new species occurred.

Remarks:The phylogenetic tree (Fig. 1) clearly places the new species in the

Caliciaceae, albeit in an isolated position. It is included in a clade thatalso includes Buellia disciformis (Fr.) Mudd, which is the type speciesof Buellia, but with a very long branch length. The genus-level phy-logeny and taxonomy of the buellioid Caliciaceae has not yet beensettled, despite recent work on the family (Marbach, 2000; Prieto and

Wedin, 2017). Species of Buellia typically have pigmented, 1-septateascospores (rarely 3-sepate) and a different ascus structure (Bacidiaor Biatora-type; Fig. 5). This type of ascus has a tholus (the area at theascus apex between the inner and outer walls) that stains dark bluewith an unstained central cone, either with (Biatora-type) or without(Bacidia-type) a darker staining wall (Rambold et al., 1994;Bungartz et al., 2007). Our new species would, therefore, be anoma-lous if placed in that genus. Submuriform ascospores are otherwiseknown in the Caliciaceae only in the genus Diplotomma Flot., which iswell-separated from our new species in our phylogeny.

The gross morphology of the new species shows a remarkablesimilarity to Rhizocarpon lavatum (Ach.) Hazsl., which occurs in simi-lar situations (damp, semi-inundated siliceous rocks) in the NorthernHemisphere (Fletcher et al., 2009) and has also been reported fromNew Zealand (Fryday, 2004), but not South Africa. However, the newspecies is readily separated from that species microscopically by thepresence of pigmented ascospores and it also differs in its ascus struc-ture and chemistry (Rhizocarpon-type and no substances in R. lava-tum).

We experienced some difficulty obtaining DNA sequence datafrom this species due to the frequent co-amplification of an

Fig. 1. Phylogeny of Caliciaceae based on maximum likelihood analysis of concatenated ITS, nrLSU, nrSSU, mtSSU, RPB1, RPB2, beta-tubulin and MCM7. Branches in bold have boot-strap support values �95; bootstrap values <70 are not displayed. Scale indicates substitutions per site. The new species is indicated with bold text.

A.M. Fryday et al. / South African Journal of Botany 132 (2020) 471�481 475

endolichenic fungus in Chaetothyriales (Eurotiomycetes). Sequencesof this fungus have been submitted to GenBank (“uncultured Chaeto-thyriales”; nrLSU, MT611532; nrSSU, MT611986). Species of Chaeto-thyriales have recently been shown to be common endolichenicinhabitants of saxicolous lichens (Muggia et al., 2016) in addition totheir previously documented role as extremophile rock-inhabitingfungi (Gueidan et al., 2008).

The ITS and nrLSU sequences for the Trebouxia photobiont areavailable on GenBank as accessions MT622498 and MT611535,respectively.

Scoliciosporum fabisporum Fryday and I. Medeiros sp. nov.Mycobank No.: MB 835880Separated from all other species of Scoliciosporum by the curved,

kidney-shaped ascospores. Also distinguished by its §flat apotheciawith a thick, slightly raised margin and its DNA sequence data.

Typus: South Africa, Mpumalanga, Ehlanzeni District, UmjindiMunicipality, Barberton, eMenzana (Badplaas) Rd, c. 1.0 km northeastof Nelshoogte, 25.8443S, 30.7795E, 1505 m., weathered ultramafic(serpentinite) rock outcrop in grassland by roadside, 30 March 2015,Fryday (11 123) and S. Siebert (PRE—holo!) (Fig. 6).

Thallus effuse, >2 cm across, grey-brown, areolate; areoles0.5�1.0 mm across with an irregular surface, edges often raised fromthe substrate and sometimes becoming §umbilicate, 0.2�0.3 mmthick; cortex absent; medulla I�. Photobiont Symbiochloris, cells9�12mm diam.

Apothecia black, lecideine, orbicular, 0.2�0.3(�0.4) mm diam.,disc, initially convex, becoming plane to slightly concave. Proper mar-gin barely apparent in young apothecia becoming more prominent inmature apothecia, poorly differentiated and barely raised,0.05�0.10 mm wide; in section cupular, 90�100 mm wide, hyalinebecoming dilute brown in the outer 12�20mm, composed of conglu-tinate, radiating branched and anastomosing hyphae, 1�2 mm wide,not widening at the surface, readily separating in K. Hymenium60�70mm tall, hyaline at the base becoming increasingly dilute aeru-ginose towards the epihymenium, at least the upper 25 mm aerugi-nose, sometimes the whole hymenium pigmented, pigment N+magenta (cinereorufa-green); paraphyses simple, thin (c. 1 mm wide)not septate, readily separating in K, not capitate; epihymenium aeru-ginose. Ascus Lecanora-type; initially cylindrical becoming slightlyclavate, 40�45 £ 10�15 mm; ascospores (0�)1-septate, fabiform,rarely straight, (10�)12.0 § 1.51(�15) £ (4�)4.6 § 0.50(�5) mm,n = 15, broadly ellipsoid with rounded apices. Hypothecium hyaline, c.70 mm thick, composed of thick (4�5 mm wide) randomly arranged,contorted hyphae, appearing almost granular.

Conidiomata not observed.Chemistry. No substances by TLC.Etymology: The specific epithet refers to the bean-shaped asco-

spores.Distribution and Ecology: The new species is known only from the

type collection where it occurred on exposed, magnesium-rich,

Fig. 2. Phylogeny of Lecanorales based on maximum likelihood analysis of concatenated ITS, nrLSU, nrSSU, mtSSU and RPB1. Branches in bold have bootstrap support values �95;bootstrap values <70 are not displayed. Scale indicates substitutions per site. The new species is indicated with bold text.

476 A.M. Fryday et al. / South African Journal of Botany 132 (2020) 471�481

ultramafic (serpentinite; Venter et al., 2018) rocks by a road-cut(Anhaeusser, 2001) in short grassland and rocky shrubland of theBarberton Montane Grassland of the Mesic Highveld Grassland Biore-gion (Mucina and Rutherford, 2006). This grassland receives meanannual precipitation of 1 194 mm and has a mean annual tempera-ture of 16.7°C. Analyzed rock samples had a Mg:Ca quotient of 24.9and total Ni concentrations of 2 200 ppm.

In 2016, we spent three weeks sampling the lichen biota of ultra-mafic rocks in Mpumalanga and did not encounter the species at anyother site. If it is an ultramafic specialist, then it would appear to bean extremely rare one. However, experience in other areas suggeststhat, unlike vascular plants, very few, if any, lichen taxa are ultramaficspecialists (Rajakaruna et al., 2012; Favero-Longo et al., 2018). Itappears more likely, therefore, that this species occurs on a widerrange of rock types and we only discovered it on ultramafic rocksbecause that was the rock type that we were sampling.

Remarks:The ITS sequence for the Symbiochloris photobiont of Scoliciospo-

rum fabisporum is available on GenBank as accession MT622499.

Our phylogenetic tree (Fig. 2) shows the new species as sister tothe Northern Hemisphere species S. intrusum (Fig. 7) in a well-sup-ported Scoliciosporaceae. The systematic position of S. intrusum haspreviously been unclear. Because of its lecideoid apothecia, it was ini-tially described by Th. Fries in the widely circumscribed genus LecideaAch. (Fries, 1867) but he soon transferred it to Catillaria A. Massal.(Fries, 1874) because of its 1-septate ascospores. Coppins and Kilias(Coppins, 1983) included it in Micarea Th. Fr., but with some hesita-tion and noted similarities with Scoliciosporum. Rambold and Triebel(Hertel, 1991; Aptroot et al., 1997) transferred the species to Car-bonea (Hertel) Hertel, before Hafellner (2004) took up Coppins andKilias’ suggestion and made the combination into Scoliciosporum. Allthese taxonomic changes were made on the basis of morphologicalcharacters alone but an initial phylogenetic analysis by Andersen andEkman (2005) with mtSSU confirmed the monophyly of Scoliciospo-rum including S. intrusum (as Micarea intrusa). However, subsequentmolecular research has suggested that S. intrusum is not con-genericwith S. umbrimum (Ach.) Arnold (the type species of Scoliciosporum).In an extensive, multi gene phylogeny, Miadlikowska et al. (2014),

Fig. 3. Burrowsia cataractae (holotype). A: Thallus and apothecia, B: Immature apothecia, C: Thallus section, D: Paraphyses (in ink, pretreated with vinegar), E: Ascus (in IKI), F: Asco-spores, G: Exciple (in K). Scale bars: A & B = 1 mm; C, F & G = 20mm; D & E = 10mm.

A.M. Fryday et al. / South African Journal of Botany 132 (2020) 471�481 477

maintained an independent Scoliciosporaceae but placed S. intrusumin the Pilocarpaceae, although they did not, for the most part, findwell-supported relationships between families in this portion of theLecanoromycetes phylogeny and the placement of S. intrusum in thePilocarpaceae was supported by some but not all of their analyses.More recently, Kraichak et al. (2018) placed S. umbrinum in theSphaerophoraceae while retaining S. intrusum in an independent Sco-liciosporaceae. However, our phylogenetic analysis places S. umbri-num, S. chlorococcum, S. intrusum, and S. fabisporum in a well-supported Scoliciosporaceae distinct from both Pilocarpaceae and

Sphaerophoraceae. The relationships between these three families,Psilolechiaceae, Ramalinaceae, and the families of suborder Lecanori-neae are still unresolved. These results will be discussed in moredetail elsewhere.

The placement of our new species in Scoliciosporum is confirmedby its morphological similarity to S. intrusum. Both species have adark dull olivaceous-brown thallus with small, black apothecia, hya-line, 0�1-septate ascospores (see Coppins, 1983, Fig.17A) and a pho-tobiont with large (to 20 mm diam.), thick walled cells. See Coppins(1983, as Micarea intrusa) and Edwards et al. (2009) for full

Fig. 4. A: Calodendrum George on Buffelskloof Nature Reserve, B: Calodendrum Falls. Burrowsia cataractae was collected from rocks at the base of the falls (photographs by JohnBurrows).

Fig. 5. Characters of typical buelluioid genera (B. gypsyensis Fryday). A, Ascus in IKIafter pretreatment with K; B, ascospores in water. Scale bars = 10mm.

478 A.M. Fryday et al. / South African Journal of Botany 132 (2020) 471�481

descriptions of S. intrusum. However, S. fabisporum differs from S.intrusum in having flat to §concave apothecia with a well-developedmargin (convex and immarginate in S. intrusum), curved ascosporesand also in the apparent lack of haustoria, which are significant char-acter of S. intrusum.

Additional specimens examined:Scoliciosporum intrusumCanada, NEWFOUNDLAND AND LABRADOR, Newfoundland, Ava-

lon Peninsula, Route 13, Witless Bay Line, about 1 km south of WitlessBay Line, south of Whale's Pond, 47.33325 �52.9537, 189 m., Sili-ceous rock, soft sedimentary outcrop in ombrotrophic Scirpus, erica-ceous, Sphagnum, Cladonia bog next to mature Abies balsamea, Piceamariana, Larix laricina, Betula papyrifera, feathermoss, Sphagnumstand, 22 June 2012, J. W. McCarthy 2264 (MSC0154938).

Finland, In TRAVASTIA, Evo, supra latus saxi subumbrosum, 1984,J. P. Norrlin s.n., (MSC01323640: Herbarium Lichenum Fenniae: Fascicu-lus IV (1875) #182 - as. Lecidea pelidna Ach.).

4. Discussion

At the end of the 19th century, Stizenberger and most otherlichenologists had a very broad concept of the genus Lecidea,

including in it all crustose species with apothecia lacking algae in themargin (Schmull et al., 2011). As such, they would have included thetwo species described here in that genus. We have checked all thedescriptions of South African species currently included in Lecideabut none resemble our new taxa.

The current South African lichen checklist (Fryday, 2016) includes71 taxa in the genus Lecidea, of which 56 are based on collectionsfrom South Africa: 20 described by Stizenberger and 36 by otherauthors (e.g., Nylander, Vainio, Zahlbruckner; see introduction). Theprevious checklist (Doidge, 1950) listed 105 species (plus 15 varie-ties) of Lecidea, but 34 of these taxa have subsequently been trans-ferred to other genera. There is no doubt that the majority of theSouth African species currently included in Lecidea belong in othergenera because the genus as currently circumscribed includes onlysaxicolous species with simple hyaline ascospores and a Lecidea-typeascus structure (Aptroot et al., 2009). As such, all corticolous and ter-ricolous species, along with many saxicolous species, will need to betransferred to other genera, but the descriptions provided in the pro-tologues are inadequate for determining the correct systematic place-ments for these species. This can only be achieved by examination ofthe type collections, which are housed in European herbaria, andeven then it is probable that many will be hard to place without DNAsequence data that will be difficult, if not impossible, to obtain fromthe 100-plus year-old type collections.

Lichens are an important component of most terrestrial ecosys-tems (see Introduction) and are also important as model systems forthe study of coevolutionary processes (O'Brien et al., 2013) andunderstanding the nature of symbiosis (Spribille et al., 2016). How-ever, their contribution to ecological processes are often overlookedor undervalued. In particular, cyanolichens — those species that con-tain a cyanobacterium as the photosynthetic partner rather than agreen chlorococcoid alga — are major contributors to nitrogen fixa-tion and cycling (Elbert et al., 2012).

The larger part of Mpumalanga forms part of the Great Escarp-ment of southern Africa, which runs all the way from Angola onthe western seaboard around the subcontinent and back north toZimbabwe/Mozambique. The diversity of species and habitatsalong the escarpment makes it an important frontier for biodiver-sity exploration (Clark et al., 2011). These new lichen taxa weredescribed from two centres of plant endemism, namely the Barber-ton and Lydenburg Centres (Schmidt et al., 2002), which are wellknown for the discovery of edaphic specialist and high-altituderestricted range species (Balkwill et al., 2011; Hankey andEdwards, 2008). The taxa described here are indicative of theundescribed lichen biodiversity of the region. During our visit toMpumalanga we collected many other lichen specimens that we

Fig. 6. Scoliciosporum fabisporum (holotype). A: Thallus and apothecia, B. Apothecia, C: Section of apothecium, D: Paraphyses (in N), E: Exciple (in K), F: Ascospores in ascus (in K), G:Ascus (in IKI). Scale bars: A = 1.0 mm; B = 0.5 mm; C = 50mm; D � G = 10mm.

A.M. Fryday et al. / South African Journal of Botany 132 (2020) 471�481 479

were unable to assign to described taxa. Work is continuing onthese and, although some are small and will have to remain as“known unknowns” (Spribille et al., 2020) until better collectionsbecome available, further species new to science are confidentlypredicted. For example, we identified at least seven distinct speciesof the genus Trapelia M. Choisy among our collections whereas

only three appear on the current checklist of South African lichens(Fryday, 2016), and one of those is a northern hemisphere speciesthat probably does not occur in South Africa. We hope that thiscontribution will prompt further investigation of the largely unex-plored, rich lichen biodiversity of the area as well as southernAfrica in general.

Fig. 7. Scoliciosporum intrusum (A �Norrlin s.n.; B & C � McCarthy 2264; both specimens in MSC). A: Thallus with apothecia, B: Section of apothecium, C: Ascospores in ascus (in N).Scale bars: A = 0.5 mm; B = 50mm; C = 10mm.

480 A.M. Fryday et al. / South African Journal of Botany 132 (2020) 471�481

Acknowledgements

We thank the South African National Research Foundation forgrant KIC14081290043 that allowed the first author to visit SouthAfrica in 2015 and the National Geographic Society for grant#9774�15 that funded the main expedition the following year. Themembers of the team extend our warmest thanks and gratitude to allthose people who made us welcome in their country during our visit,in particular John and Sandie Burrows at Buffelskloof Nature Reserve.John is also thanked for providing the photographs used in Fig. 4.Ma�ns Svensson (Uppsala) is thanked for help with the molecularanalysis of Scoliciosporum. We also thank Arnold Frisby (Pretoria) forlogistic support. The Department of Biology at Duke University pro-vided financial support for molecular work. This research is based, inpart, upon work supported by the National Science Foundation Grad-uate Research Fellowship Program under grant number DGE1644868. Any opinions, findings, and conclusions or recommenda-tions expressed here are those of the authors and do not necessarilyreflect the views of the National Science Foundation.

References

Ahti, T., Mayrhofer, H., Schultz, M., Tehler, A., Fryday, A.M., 2016. Additions, deletionsand other changes to the lichen checklist of South Africa. Bothalia 46 (1), a2065.https://doi.org/10.4102/abc.v46i1.2065.

Almborn, O., 1966. Revision of some lichen genera in southern Africa I. BotaniskaNotiser 119, 70–112.

Almborn, O., 1987. Lichens at high altitudes in southern Africa. Bibliotheca Lichenolog-ica 25, 401–417.

Andersen, H.L., Ekman, S., 2005. Disintegration of the Micareaceae (lichenized Ascomy-cota): a molecular phylogeny based on mitochondrial rDNA sequences. Mycologi-cal Research 109, 21–30.

Anhaeusser, C.R., 2001. The anatomy of an extrusive-intrusive Archaean mafic-ultra-mafic sequence: the Nelshoogte Schist Belt and Stolzburg Layered Ultramafic Com-plex, Barberton Greenstone Belt, South Africa. South African Journal of Geology104 (2), 167–204. https://doi.org/10.2113/1040167.

Aptroot, A., Diederich, P., S�erusiaux, E., Sipman, H.J.M., 1997. Lichens and LichenicolousFungi from New Guinea. Bibliotheca Lichenologica 64, 1–220.

Aptroot, A., Gilbert, O.L., Hawksworth, D.L., Coppins, B.J., 2009. Lecidea Ach. (1803).In: Smith, C.W., Aptroot, A., Coppins, B.J., Fletcher, A., Gilbert, O.L., James, P.W.,Wolseley, P.A. (Eds.), The Lichens of Great Britain and Ireland. British Lichen Soci-ety, London, pp. 502–519.

Aptroot, A., Maphangwa, K., Zedda, L., Tekere, M., Alvarado, P., Sipman, H., 2019. Thephylogenetic position of Culbersonia is in the Caliciaceae (lichenized ascomycetes).The Lichenologist 51, 187–191. https://doi.org/10.1017/S0024282919000033.

Asplund, J., Wardle, D.A., 2013. The impact of secondary compounds and functionalcharacteristics on lichen palatability and decomposition. Journal of Ecology 101,689–700.

Asplund, J., Wardle, D.A., 2017. How lichens impact on terrestrial community and eco-system properties. Biological Reviews 92 (3), 1720–1738.

Balkwill, K., Campbell-Young, G.J., Fish, L., Munday, J., Frean, M.L., Stalmans, M., 2011. Anew species of Sartidia (Graminae), endemic to ultramafic soils. South African Jour-nal of Botany 77 (3), 598–607.

Berger, S.A., Krompass, D., Stamatakis, A., 2011. Performance, accuracy, and web serverfor evolutionary placement of short sequence reads under maximum likelihood.Systematic Biology 60, 291–302.

Bungartz, F., Nordin, A., Grube, U., 2007. Buellia De Not.. In: Nash, III, T.H., Gries, C.,Bungartz, F. (Eds.), Lichen Flora of the Greater Sonoran Desert Region. 3, LichensUnlimited, Arizona State University, Tempe, pp. 113–179.

Carbone, I., White, J.B., Miadlikowska, J., Arnold, A.E., Miller, M.A., Kauff, F., U'Ren, J.M.,May, G., Lutzoni, F., 2016. T-BAS: Tree-Based Alignment Selector toolkit for phylo-genetic-based placement, alignment downloads and metadata visualization: Anexample with the Pezizomycotina tree of life. Bioinformatics 33, 1160–1168.

Carbone, I., White, J.B., Miadlikowska, J., Arnold, A.E., Miller, M.A., Magain, N., U’Ren, J.M.,Lutzoni, F., 2019. T-BAS version 2.1: Tree-Based Alignment Selector toolkit for evolu-tionary placement and viewing of alignments and metadata on curated and customtrees. Microbiological Resource Announcements 8, e00328 -19.

Clark, V.R., Barker, N.P., Mucina, L., 2011. The Great Escarpment of southern Africa: a newfrontier for biodiversity exploration. Biodiversity and Conservation 20 (12), 2543.

Coppins, B.J., 1983. A taxonomic study of the lichen genus Micarea in Europe. Bulletinof the British Museum (Natural History). Botany Series 11 (2), 17–214.

Crombie, J.M., 1876. Lichenes capenses: — An Enumeration of the Lichens collected atthe Cape of Good Hope by the Rev. A. E. Eaton during the Venus-Transit Expeditionin 1874. Journal of the Linnean Society, Botany 15, 165–180.

Crous, P.W., Rong, I.H., Wood, A., Lee, S., Glen, H., Botha, W., Slippers, B., de Beer, W.Z.,Wingfield, M.J., Hawksworth, D.L., 2006. How many species of fungi are there atthe tip of Africa? Studies in Mycology 55, 13–33. https://doi.org/10.3114/sim.55.1.13.

Doidge, E.M., 1950. The South African fungi and lichens to the end of 1945. Bothalia 5,1–1094.

Edwards, B.W., James, P.W., Purvis, O.W., 2009. Scoliciosporum A. Massal. (1852).In: Smith, C.W., Aptroot, A., Coppins, B.J., Fletcher, A., Gilbert, O.L., James, P.W.,Wolseley, P.A. (Eds.), The Lichens of Great Britain and Ireland. British Lichen Soci-ety, London, pp. 839–841.

Elbert, W., Weber, B., Burrows, S., Steinkamp, J., B€udel, B., Andreae, M.O., P€oschl, U.,2012. Contribution of cryptogamic covers to the global cycles of carbon and nitro-gen. Nature Geoscience 5 (7), 459–462.

Favero-Longo, S.E., Matteucci, E., Giordani, P., Paukov, A., Rajakaruna, N., 2018. Diver-sity and functional traits of lichens in ultramafic areas: a literature-based world-wide analysis integrated by field data at the regional scale. Ecological Research 33,593–608.

Fletcher, A., Gilbert, O.L., Clayden, S., Fryday, A.M., 2009. Rhizocarpon Ramond ex DC.(1805). In: Smith, C.W., Aptroot, A., Coppins, B.J., Fletcher, A., Gilbert, O.L.,James, P.W., Wolseley, P.A. (Eds.), The Lichens of Great Britain and Ireland. BritishLichen Society, London, pp. 792–808.

Fries, T.M., 1867. Nya skandinaviska laf-arter No. 91-100. Botaniska Notiser 1867, 151–155.

Fries, T.M., 1874. Lichenographia Scandinavica sive dispositio lichenum in Dania, Sue-cia, Norvegia, Fennia, Lapponia Rossica hactus collectorum. Vol. I Archilichenes dis-cocarpos continens. Pars II. Berling, Upsaliae.

Fryday, A.M., 2004. New species and records of lichenized fungi from Campbell Islandand the Auckland Islands, New Zealand. Bibliotheca Lichenologica 88, 127–146.

Fryday, A.M., 2015. A new checklist of lichenised, lichenicolous and allied fungireported from South Africa. Bothalia 45 (1) Art. #148, 4 pages http://doi.org/10.4102/abc.v45i1.148.

Fryday, A.M., 2016. A new checklist of lichenised, lichenicolous and allied fungireported from South Africa’, viewed 8 January 2020. http://herbarium.msu.edu/lichen/South_Africa/SA_List-latest.html.

Gardes, M., Bruns, T.D., 1993. ITS primers with enhanced specificity for basidiomy-cetes—Application to the identification of mycorrhizae and rusts. Molecular Ecol-ogy 2, 113–118.

Gargas, A., Taylor, J., 1992. Polymerase chain reaction (PCR) primers for amplifying andsequencing nuclear 18s rDNA from lichenized fungi. Mycologia 84 (4), 589–592.

Gueidan, C., Ruibal Villase~nor, C., de Hoog, G.S., Gorbushina, A.A., Untereiner, W.A.,Lutzoni, F., 2008. A rock-inhabiting ancestor for mutualistic and pathogen-rich fun-gal lineages. Studies in Mycology 61, 111–119.

Hankey, A.J., Edwards, T.J., 2008. Ledebouria mokobulanensis A.J. Hankey and T.J.Edwards (Hyacinthaceae) a new species from the high altitude grasslands of Mpu-malanga. South African Journal of Botany 74 (2), 214–217.

Hertel, H., 1991. Lecidea in der Arktis III (lecideoide Flechten; Lecanorales). Mitteilun-gen der Botanischen Staatssammlung M€unchen 30, 297–333.

Hofstetter, V., Miadlikowska, J., Kauff, F., Lutzoni, F., 2007. Phylogenetic comparison of pro-tein-coding versus ribosomal RNA-coding sequence data: A case study of the Lecanor-omycetes (Ascomycota). Molecular Phylogenetics and Evolution 44, 412–426.

Katoh, K., Rozewicki, J., Yamada, K.D., 2017. MAFFT online service: multiple sequencealignment, interactive sequence choice and visualization. Briefings in Bioinformat-ics 20 (4), 1160–1166. https://doi.org/10.1093/bib/bbx108.

Kauff, F., Lutzoni, F., 2002. Phylogeny of the Gyalectales and Ostropales (Ascomycota,Fungi): Among and within order relationships based on nuclear ribosomal RNAsmall and large subunits. Molecular Phylogenetics and Evolution 25, 138–156.

Kondratyuk, S.Y., K€arnefelt, I., Thell, A., Elix, J.A., Kim, J.A., Kondratiuk, A.S., Hur, J.-.S.,2015. Ovealmbornia reginae (Teloschistaceae, Ascomycetes), a New XanthorioidLichen from South Africa. Herzogia 28, 465–472.

Knops, J.M.H., Nash, III, T.H., Schlesinger, W.H., 1996. The influence of epiphytic lichens onthe nutrient cycling of an oakwoodland. Ecological Monographs 66 (2), 159–179.

Kraichak, E., Huang, J.P., Nelsen, M., Leavitt, S.D., Lumbsch, H.T., 2018. A revised classifi-cation of orders and families in the two major subclasses of Lecanoromycetes(Ascomycota) based on a temporal approach. Botanical Journal of the Linnean Soci-ety 188 (3), 233–249.

Kroken, S., Taylor, J.W., 2000. Phylogenetic species, reproductive mode, and specificityof the green alga Trebouxia forming lichens with the fungal genus Letharia. TheBryologist 103, 645–660.

Kuraku, S., Zmasek, C.M., Nishimura, O., Katoh, K., 2013. aLeaves facilitates on-demandexploration of metazoan gene family trees on MAFFT sequence alignment serverwith enhanced interactivity. Nucleic Acids Research 41 (W1), W22–W28.

Lanfear, R., Frandsen, P.B., Wright, A.M., Senfeld, T., Calcott, B., 2016. PartitionFinder 2:new methods for selecting partitioned models of evolution for molecular and mor-phological phylogenetic analyses. Molecular Biology and Evolution 34, 772–773.

Leavitt, S.D., Kirika, P.M., Amo De Paz, G., Huang, J.-.P., Hur, J.-.S., Elix, J.A., Grewe, F.,Divakar, P.K., Lumbsch, H.T., 2018a. Assessing phylogeny and historical biogeogra-phy of the largest genus of lichen-forming fungi, Xanthoparmelia (Parmeliaceae,Ascomycota). The Lichenologist 50, 299–312.

Leavitt, S.D., Westberg, M., Nelsen, M.P., Elix, J.A., Timdal, E., Sohrabi, M., St. Clair, L.L.,Williams, L., Wedin, M., Lumbsch, H.T., 2018b. Multiple, distinct intercontinentallineages but isolation of Australian populations in a cosmopolitan lichen-formingfungal taxon, Psora decipiens (Psoraceae, Ascomycota). Frontiers in Microbiology 9,283. https://doi.org/10.3389/fmicb.2018.00283.

Lendemer, J.C., Harris, R.C., Tripp, E.A., 2013. The lichens and allied fungi of Great SmokyMountains National Park. Memoires of the New York Botanical Garden 104, 1–152.

Liu, Y.J., Whelen, S., Hall, B.D., 1999. Phylogenetic relationships among ascomycetes:evidence from an RNA polymerase II subunit. Molecular Biology and Evolution 16(12), 1799–1808.

L€otter, M.C., Mucina, L., Witkowski, E.T.F., 2014. Classification of the indigenous forestsof Mpumalanga Province, South Africa. South African Journal of Botany 90, 37–51.

Marbach, B., 2000. Corticole und lignicole Arten der Flechtengattung Buellia-sensu latoin den Subtropen und Tropen. Bibliotheca Lichenologica 74, 1–384.

Matheny, P.B., Liu, Y.J., Ammirati, J.F., Hall, B.D., 2002. Using RPB1 sequences to improvephylogenetic inference among mushrooms (Inocybe, Agaricales). American Journalof Botany 89, 688–698.

Meyer, B., Printzen, C., 2000. Proposal for a standardized nomenclature and characteri-zation of insoluble lichen pigments.. The Lichenologist 32, 571–583.

Miadlikowska, J., Kauff, F., H€ognabba, F., Oliver, J.C., Moln�ar, K., Fraker, E., Gaya, E.,Hafellner, J., Hofstetter, V., Gueidan, C., Ot�alora, M.A.G., Hodkinson, B., Kukwa, M.,L€ucking, R., Bj€ork, C., Sipman, H.J.M., Burgaz, A.R., Thell, A., Passo, A., Myllys, L.,Goward, T., Fern�andez-Brimem, S., Hestmark, G., Lendemer, J., Lumbsch, H.T.,Schmull, M., Schoch, C.L., S�erusiaux, E., Maddison, D.R., Arnold, A.E., Lutzoni, F.,Stenroos, S., 2014. A multigene phylogenetic synthesis for the class Lecanoromy-cetes (Ascomycota): 1307 fungi representing 1139 infrageneric taxa, 317 generaand 66 families. Molecular Phylogenetics and Evolution 79, 132–168.

Miller, M.A., Pfeiffer, W., Schwartz, T., 2010. Creating the CIPRES Science Gateway forinference of large phylogenetic trees. In: Proceedings of the Gateway ComputingEnvironments Workshop, pp. 1–8.

Miller, M.A., Schwartz, T., Pickett, B.E., He, S., Klem, E.B., Scheuermann, R.H.,Passarotti, M., Kaufman, S., O’Leary, M.A., 2015. A RESTful API for access to phyloge-netic tools via the CIPRES science gateway. Evolution and Bioinformatics 11, 43–48.

Mucina, L., Rutherford, M.C., 2006. The Vegetation of South Africa, Lesotho and Swazi-land (Strelitzia 19). South African National Biodiversity Institute, Pretoria.

Muggia, L., Fleischhacker, A., Kopun, T., Grube, M., 2016. Extremotolerant fungi from alpinerock lichens and their phylogenetic relationships. Fungal Diversity 76, 119–142.

Nylander, W., 1869. Note sur les lichens de Port-Natal. Bulletin de la Societe Linneennede Normandie 3, 4–15.

O'Brien, H.E., Miadlikowska, J., Lutzoni, F., 2013. Assessing population structure andhost specialization in lichenized cyanobacteria. New Phytologist 198 (2), 557–566.

Orange, A., James, P.W., White, F.J., 2001. Microchemical Methods for the Identificationof Lichens.. British Lichen Society, London.

Pattengale, N.D., Alipour, M., Bininda-Emonds, O.R., Moret, B.M., Stamatakis, A., 2009.Howmany bootstrap replicates are necessary? In: Batzoglou, S. (Ed.), Annual Inter-national Conference On Research in Computational Molecular Biology. Springer,Berlin, Heidelberg, pp. 184–200.

Prieto, M., Wedin, M., 2017. Phylogeny, taxonomy and diversification events in the Cal-iciaceae. Fungal Diversity 82, 221–238.

Rambold, G., Mayrhofer, H., Matzer, M., 1994. On the ascus types in the Physciaceae(Lecanorales). Plant Systematics and Evolution 192, 31–40.

Rajakaruna, N., Knudsen, K., Fryday, A.M., O’Dell, R.E., Pope, N., Olday, F.C.,Woolhouse, S., 2012. Investigation of the importance of rock chemistry for saxico-lous lichen communities of the New Idria serpentinite mass, San Benito County,California, USA. The Lichenologist 44, 695–714.

Rehner, S.A., Samuels, G.J., 1994. Taxonomy and phylogeny of Gliocladium analyzedfrom nuclear large subunit ribosomal DNA sequences. Mycological Research 98,625–634.

Schmidt, E., Lotter, M., McCleland, W., 2002. Trees and Shrubs of Mpumalanga andKruger National Park. Jacana, Johannesburg.

Schmull, M., Miadlikowska, J., Pelzer, M., Stocker-W€org€otter, E., Hofstetter, V.,Fraker, E., Hodkinson, B.P., Reeb, V., Kukwa, M., Lumbsch, H.T., Kauff, F., Lutzoni, F.,2011. Phylogenetic affiliations of members of the heterogeneous lichen-formingfungi of the genus Lecidea sensu Zahlbruckner (Lecanoromycetes, Ascomycota).Mycologia 103, 983–1003.

Schnitzler, J., Barraclough, T.G., Boatwright, J.S., Goldblatt, P., Manning, J.C.,Powell, M.P., Rebelo, A., Savolainen, V., 2011. Causes of plant diversification in theCape biodiversity hotspot of South Africa. Systematic Biology 60, 343–357. http://dx.doi.org/10.1093/sysbio/syr006.

Schultz, M., Zedda, L., Rambold, G., 2009. New records of lichen taxa from Namibia andSouth Africa. Bibliotheca Lichenologica 99, 315–334.

Slack, N.G., 1988. The ecological importance of lichens and bryophytes. BibliothecaLichenologica 30, 23–53.

Smith, C.W., Aptroot, A., Coppins, B.J., Fletcher, A., Gilbert, O.L., James, P.W., Wolseley, P.A. (Eds), The Lichens of Great Britain and Ireland. British Lichen Society, London.

Spribille, T., Tuovinen, V., Resl, P., Vanderpool, D., Wolinski, H., Aime, M.C.,Schneider, K., Stabentheiner, E., Toome-Heller, M., Thor, G., Mayrhofer, H.,Johannesson, H., McCutcheon, J.P., 2016. Basidiomycete yeasts in the cortex ofascomycete macrolichens. Science 353 (6298), 488–492.

Spribille, T., Fryday, A.M., P�erez-Ortega, S., Svensson, M., Tønsberg, T., Ekman, S.,Holien, H., Resl, P., Schneider, K., Stabentheiner, E., Th€us, H., Vondr�ak, J.,Sharman, L., 2020. Lichens and associated fungi from Glacier Bay National Park,Alaska. The Lichenologist 52 (2), 61–181 (in press).

Stamatakis, A., 2014. RAxML version 8: a tool for phylogenetic analysis and post-analy-sis of large phylogenies.. Bioinformatics 30 (9), 1312–1313.

Stizenberger, E., 1890. Lichenaea Africana. Bericht €uber die Th€atigkeit der St. Gallischennaturwissenschaftliche Gesellschaft, 1888-89, pp. 105–249.

Stizenberger, E., 1891. Lichenaea Africana. Bericht €uber die Th€atigkeit der St. Gallischennaturwissenschaftliche Gesellschaft 1889-90, 133–268.

Vainio, E., 1926. Lichenes Africani novi. Annales Universitatis Fennicae Abo€ensis ser. A2 (3), 1–33.

Venter, A., Siebert, S.J., Rajakaruna, N., Barnard, S., Levanets, A., Ismail, A., Allam, M.,Peterson, B., Sanko, T., 2018. Biological crusts of serpentine and non�serpentinesoils from the Barberton Greenstone Belt of South Africa. Ecological Research 33(3), 629–640.

Vilgalys, R., Hester, M., 1990. Rapid genetic identification and mapping of enzymati-cally amplified ribosomal DNA from several Cryptococcus species. Journal of Bacte-riology 172, 4238–4246.

White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and direct sequencing of fun-gal ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand, D.H.,Sninsky, J.J., White, T.J. (Eds.), PCR protocols: A guide to Methods and Applications.Academic Press, London, pp. 315–322.

Zahlbruckner, A., 1906. Die Flechten der Deutschen S€udpolar-Expedition 1901-1903.Deutsche S€udpolar-Expedition 1901-03 VIII, Botanik, 19–55.

Zahlbruckner, A., 1926. Afrikanische Flechten (Lichenes). Englers BotanischeJahrb€ucher 60, 468–552.

Zahlbruckner, A., 1932. Lichenes in Africa lecti. Annales de Cryptogamie Exotique 5,198–275.

Zahlbruckner, A., 1936. Neue Flechten, XII. Annales of Mycology 34, 159–179.Zhang, Z., Schwartz, S., Wagner, L., Miller, W., 2000. A greedy algorithm for aligning

DNA sequences. Journal of Computational Biology 7, 203–214.Zoller, S., Scheidegger, C., Sperisen, C., 1999. PCR primers for the amplification of mito-

chondrial small subunit ribosomal DNA of lichen-forming ascomycetes. TheLichenologist 31, 511–516.